Dicer is a nuclease involved in processing double-stranded RNA (dsRNA) into small RNAs called siRNA and miRNA. These small RNAs are involved in regulating the expression of numerous genes, in diverse organisms, including humans. The proposal will focus on aspects of Dicer that are largely undefined, such as the function of its helicase domain and its role in silencing by endogenous siRNA (endo-siRNA). Mutations will be introduced into the helicase domain of Dicer, and effects monitored in vivo using the model organism C. elegans, and in vitro using biochemical techniques. The hypothesis that Dicer's helicase domain allows it to function processively will be tested. The laboratory's recent results indicate animals with mutations in the helicase domain are devoid of certain endo-siRNA and accumulate their precursors. The latter will allow the first characterization of endo-siRNA precursors. The proposed research will advance our understanding of Dicer, an enzyme that is absolutely essential for life.